DNASE MEDIA

Cat. no. G24 DNase with Toluidine Blue, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

Hardy Diagnostics DNase with Toluidine Blue Agar is recommended for the detection of DNase in gram-negative bacteria, especially Serratia spp.

SUMMARY

Jeffries, Holtman, and Guse, in 1957, reported the development of a medium which detected deoxyribonuclease activity of microorganisms.(7) When plates, containing 18-24 hours of bacterial growth, were flooded with 0.1N hydrochloric acid (HCl), organisms possessing deoxyribonuclease (DNase) degraded the DNA into nucleotide fractions resulting in a clear zone around the colonies. When applied to organisms that do not produce DNase, HCl combines with nucleate salts to yield free nucleic acid, thereby producing an opaque appearance in the medium.

DNase with Toluidine Blue O, developed by Schreier in 1969, is a modification of the formula developed by Jeffries, Holtman, and Guse.(7,8) The basal medium is composed of casein and soy peptones which supply necessary nutrients. Sodium chloride is added to maintain osmotic equilibrium. DNA is incorporated into the medium to detect organisms which possess the enzyme deoxyribonuclease. Toluidine blue O serves as the color indicator. The addition of toluidine blue eliminates the need to add HCl to the plate.

The complexing of toluidine blue O with DNA produces a blue color in the uninoculated medium. Organisms which depolymerize DNA result in the formation of a dye, oligonucleotide, and mononucleotide complex. Metachromatic properties of the indicator thereby produce a visible bright rose-pink color in the surrounding area of the organisms possessing the DNase enzyme.

Because toluidine blue may be inhibitory to some gram-positive organisms, it is recommended for the detection of DNase in gram-negative microorganisms.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 15.0gm
Papaic Digest of Soybean Meal 5.0gm
Sodium Chloride 5.0gm
Deoxyribonucleic Acid 2.0gm
Agar 15.0gm
Toluidine Blue O 0.075gm

Final pH 7.3 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Method of Use:

1. Allow the agar plates or tubes to warm to room temperature before use.

2. Using a single heavy streak or a spot inoculum of approximately 5mm diameter, inoculate the medium with an 18-24 hour old isolated colony of the organism to be tested. Several cultures may be tested on one plate.

3. Incubate at 35ºC. for 18-24 hours in an aerobic atmosphere.

4. Following incubation:

DNase with Toluidine Blue: Observe for a rose-pink zone of color around growth, which is indicative of DNase producers.

INTERPRETATION OF RESULTS

DNase with Toluidine Blue: A positive reaction is indicated by the development of a rose-pink color around the inoculum. A negative reaction is indicated by growth with no change in color (blue).

LIMITATIONS

The test organisms must be in pure culture and 18-24 hours old.

DNase with Toluidine Blue may be inhibitory to some strains of gram-positive bacteria, particularly staphylococci. When testing for Branhamella ( Moraxella ) catarrhalis , a very heavy inoculum must be used.

An inoculum that is too broad may result in complete decolorization of the media, due to the reduction of the dye. If this occurs, the test results must be repeated.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, Hydrochloric Acid, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
DNase with Toluidine Blue:
Serratia marcescens
ATCC ® 13880
E 24-48hr 35°C Aerobic Growth; pink zone around colonies, must use transmitted light to read, may take up to 48 hours for color change
Branhamella
( Moraxella ) catarrhalis
ATCC ® 25240

E 24-48hr 35°C CO 2 ** Growth; pink zone around colonies, must use transmitted light to read, may take up to 48 hours for color change
Staphylococcus aureus
ATCC ® 25923
E 24-48hr 35°C Aerobic Growth; pink zone around colonies, must use transmitted light to read, may take up to 48 hours for color change
Escherichia coli
ATCC ® 25922
E 24-48hr 35°C Aerobic Growth; no color change
Staphylococcus epidermidis
ATCC ® 12228
E 24-48hr 35°C Aerobic Growth; no color change

Note: Plates are inoculated with a single heavy streak.

User Quality Control

PHYSICAL APPEARANCE

DNase with Toluidine Blue should appear clear, slightly opalescent, and blue in color.

DNase Media

Uninoculated plate of DNase Media with Toluidine Blue (Cat. no. G24).

S. marcescens growing on DNase w/ Toluidine Blue

Serratia marcescens (ATCC ® 13880) growing on DNase with Toluidine Blue (Cat. no. G24). Incubated aerobically for 48 hours at 35ºC.

M. catarrhalis growing on DNase w/ Toluidine Blue

Moraxella catarrhalis (ATCC ® 25240) growing on DNase with Toluidine Blue (Cat. no. G24). Incubated in CO 2 for 48 hours at 35ºC.

S. aureus growing on DNase w/ Toluidine Blue

Staphylococcus aureus (ATCC ® 25923) growing on DNase with Toluidine Blue (Cat. no. G24). Incubated aerobically for 48 hours at 35ºC.

S. epidermidis growing on DNase w/ Toludine Blue

Staphylococcus epidermidis (ATCC ® 12228) growing on DNase with Toluidine Blue (Cat. no. G24). Incubated aerobically for 24 hours at 35ºC.

E. coli growing on DNase w/ Toluidine Blue

Escherichia coli (ATCC ® 25922) growing on DNase with Toluidine Blue (Cat. no. G24). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. MacFaddin, J.F. 1985. Media for Isolation , Cultivation , Identification , Maintenance of Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.

5. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

6. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

7. Jeffries, Holtman and Guse. 1957. J. Bacteriol. ; 73:590.

8. Schreier, J.B. 1969. Am. J. Clin. Pathol. ; 51:711-716.


ATCC is a registered trademark of the American Type Culture Collection.

022316gr